Jinyoung Kim

Select Nutrients in the Ovine Uterine Lumen. VII. Effects of Arginine, Leucine, Glutamine, and Glucose on Trophectoderm Cell Signaling, Proliferation, and Migration

Histotroph is required for survival and development of ovine conceptuses (embryo and extraembryonic membranes). Results from our laboratory indicate that arginine (Arg), leucine (Leu), glutamine (Gln), and glucose increase in the uterine lumen between Days 10 and 15 of pregnancy, coincident with increases in expression of amino acid and glucose transporters by uterine epithelia as well as trophectoderm and yolk sac of conceptuses and elongation of the conceptus trophectoderm. Therefore, we hypothesized that Arg, Leu, Gln, and glucose have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary ovine trophectoderm (oTr) cells isolated from Day 15 conceptuses were serum-starved for 24 h in a customized medium, deprived of select nutrients, and then treated with either Arg, Leu, Gln, or glucose. Western blot analyses of whole oTr cell extracts revealed that Arg, Leu, and glucose, but not Gln, increased phosphorylated AKT1 by 2.8-, 2.5-, and 1.8-fold, respectively, within 15 min, and the increase was maintained to 60 min. Arg, Leu, and glucose also stimulated increases in phosphorylated ribosomal protein S6K (pRPS6K) by 4.2-, 4.7-, and 2.3-fold, respectively, within 15 min, as well as increases in phosphorylated ribosomal protein S6 (pRPS6) between 0 and 30 min posttreatment, that were sustained to 60 min. When oTr cells were treated with Arg, pRPS6K protein increased in nuclei, but this was not observed in nuclei of oTr cells treated with Leu and glucose. Immunocytochemical analyses also revealed abundant amounts of pRPS6 protein in the cytoplasm of oTr cells treated with Arg, Leu, and glucose. Furthermore, Arg and Leu increased proliferation and migration of oTr cells. Collectively, these results indicate that Arg, Leu, and glucose, but not Gln, in histotroph coordinately activate AKT1-mechanistic target of rapamycin and RPS6K-RPS6 cell signaling pathways to stimulate hypertrophy, hyperplasia, and migration of oTr cells.

Select Nutrients in the Ovine Uterine Lumen. VIII. Arginine Stimulates Proliferation of Ovine Trophectoderm Cells Through MTOR-RPS6K-RPS6 Signaling Cascade and Synthesis of Nitric Oxide and Polyamines

During the peri-implantation period in sheep, l-arginine (l-Arg) in the uterine lumen is an essential substrate for the synthesis of nitric oxide (NO), by nitric oxide synthase (NOS), and polyamines, via arginase and ornithine decarboxylase, that are required for survival and development of ovine conceptuses (embryo and its extraembryonic membranes). l-Arginine can stimulate hypertrophy, hyperplasia, and differentiation of the ovine conceptus trophectoderm; however, the responsible signal transduction cascade has not been determined. Therefore, this study examined possible signaling pathways mediated by l-Arg, as well as the effects of two NO donors (S-nitroso-N-acetyl-dl-penicillamine and diethylenetriamine) and putrescine (precursor for spermidine and spermine) on oTr cell proliferation. Further, the inhibition of these effects by l-NAME (l-nitro-arginine methyl ester, an inhibitor of NOS) and nor-NOHA (N-omega-hydroxy-nor-arginine, an inhibitor of arginase) was assessed. l-Arginine treatment increased the abundance of phosphorylated MTOR, RPS6K, and EIF4EBP1 in oTr cells. Consistent with activation of these cell-signaling molecules, l-Arg increased protein synthesis and reduced protein degradation in oTr cells. Both NO and polyamines enhanced cell proliferation in a dose-dependent manner. The effects of l-Arg were partially inhibited by both l-NAME and nor-NOHA. These results indicate that l-Arg enhances production of polyamines and NO and activates the MTOR/FRAP1-RPS6K-RPS6 signaling pathway to stimulate proliferation and migration of oTr cells.

Insulin-Like Growth Factor II Activates Phosphatidylinositol 3-Kinase-Protooncogenic Protein Kinase 1 and Mitogen-Activated Protein Kinase Cell Signaling Pathways, and Stimulates Migration of Ovine Trophectoderm Cells

IGF-II, a potent stimulator of cellular proliferation, differentiation, and development, regulates uterine function and conceptus growth in several species. In situ hybridization analyses found that IGF-II mRNA was most abundant in the caruncular endometrial stroma of both cyclical and pregnant ewes. In the intercaruncular endometrium, IGF-II mRNA transitioned from stroma to luminal epithelium between d 14 and 20 of pregnancy. IGF-II mRNA was present in all cells of the conceptus but was particularly abundant in the yolk sac. Immunohistochemical analyses revealed that phosphorylated (p)-protooncogenic protein kinase 1, p-ribosomal protein S6 kinase, p-ERK1/2, and p-P38 MAPK proteins were present at low levels in a majority of endometrial cells but were most abundant in the nuclei of endometrial luminal epithelium and conceptus trophectoderm of pregnant ewes. In mononuclear trophectoderm cells isolated from d-15 conceptuses, IGF-II increased the abundance of p-pyruvate dehydrogenase kinase 1, p-protooncogenic protein kinase 1, p-glycogen synthase kinase 3B, p-FK506 binding protein 12-rapamycin associated protein 1, and p-ribosomal protein S6 kinase protein within 15 min, and the increase was maintained for 90 min. IGF-II also elicited a rapid increase in p-ERK1/2 and p-P38 MAPK proteins that was maximal at 15 or 30 min posttreatment. Moreover, IGF-II increased migration of trophectoderm cells. Collectively, these results support the hypothesis that IGF-II coordinately activates multiple cell signaling pathways critical to survival, growth, and differentiation of the ovine conceptus during early pregnancy.

Secreted phosphoprotein 1 binds integrins to initiate multiple cell signaling pathways, including FRAP1/mTOR, to support attachment and force-generated migration of trophectoderm cells

Attachment and migration of trophectoderm (Tr) cells, hallmarks of blastocyst implantation in mammals, are unique uterine events. Secreted phosphoprotein 1 (SPP1) in the uterus binds integrins on conceptus Tr and uterine luminal epithelium (LE), affecting cell-cell and cell-matrix interactions. The signal transduction pathways activated by SPP1 and integrins in conceptuses have not been elucidated. Results of this study demonstrate that SPP1 binds alphavbeta3 and alpha5beta1 integrins to induce focal adhesion assembly, a prerequisite for adhesion and migration of Tr, through activation of: 1) P70S6K via crosstalk between FRAP1/mTOR and MAPK pathways; 2) mTOR, PI3K, MAPK3/MAPK1 (Erk1/2) and MAPK14 (p38) signaling to stimulate Tr cell migration; and 3) focal adhesion assembly and myosin II motor activity to induce migration of Tr cells. These cell signaling pathways, acting in concert, mediate adhesion, migration and cytoskeletal remodeling of Tr cells essential for expansion and elongation of conceptuses and attachment to uterine LE for implantation.

Select Nutrients in the Ovine Uterine Lumen. IX. Differential Effects of Arginine, Leucine, Glutamine, and Glucose on Interferon Tau, Ornithine Decarboxylase, and Nitric Oxide Synthase in the Ovine Conceptus

Nutrients are primary requirements for development of conceptuses (embryo and extraembryonic membranes), including protein synthesis. We have shown that arginine (Arg), leucine (Leu), and glucose stimulate protein synthesis through phosphorylation of MTOR signaling molecules, thereby increasing proliferation of ovine trophectoderm cells. This study determined whether Arg, Leu, glutamine (Gln), and glucose influence gene expression and protein synthesis in explant cultures of ovine conceptuses recovered from ewes on Day 16 of pregnancy. Conceptuses were deprived of select nutrients and then cultured with either Arg, Leu, Gln, or glucose for 18 h, after which they were analyzed for abundance of MTOR, RPS6K, RPS6, EIF4EBP1 (also known as 4EBP1), IFNT, NOS2, NOS3, GCH1, and ODC1 mRNAs and proteins. Levels of MTOR, RPS6K, RPS6, and EIF4EBP1 mRNAs were not affected by treatment with any of the select nutrients. Similarly, expression of IFNT, NOS2, NOS3, and ODC1 mRNAs were not different. Interestingly, GCH1 mRNA levels increased in response to Arg treatment. Importantly, Arg, Leu, Gln, and glucose increased the abundance of phosphorylated MTOR, RPS6K, RPS6, and EIF4EBP1 proteins as well as NOS and ODC1 proteins, but only Arg increased the abundance of IFNT protein. These findings indicate that Arg, Leu, Gln, and glucose stimulate translation of mRNAs to increase synthesis of proteins through phosphorylation and activation of components of the MTOR signaling pathway. Increases in abundance of IFNT protein (the pregnancy recognition signal), NOS2, NOS3 and GCH1 for conversion of Arg to nitric oxide, and ODC1 for synthesis of polyamines are all important for growth and development of the ovine conceptus during pregnancy.

Insulin-Like Growth Factor Binding Protein-1 in the Ruminant Uterus: Potential Endometrial Marker and Regulator of Conceptus Elongation

Establishment of pregnancy in ruminants requires conceptus elongation and production of interferon-tau (IFNT), the pregnancy recognition signal that maintains ovarian progesterone (P4) production. These studies determined temporal and spatial alterations in IGF binding protein (IGFBP)-1 and IGFBP3 in the ovine and bovine uterus; effects of P4 and IFNT on their expression in the ovine uterus; and effects of IGFBP1 on ovine trophectoderm cell proliferation, migration, and attachment. IGFBP1 and IGFBP3 were studied because they are the only IGFBPs specifically expressed by the endometrial luminal epithelia in sheep. In sheep, IGFBP1 and IGFBP3 expression was coordinate with the period of conceptus elongation, whereas only IGFBP1 expression was coordinate with conceptus elongation in cattle. IGFBP1 mRNA in the ovine endometria was between 5- and 29-fold more abundant between d 12 and 16 of pregnancy compared with the estrous cycle and greater on d 16 of pregnancy than nonpregnancy in the bovine uterus. In sheep, P4 induced and IFNT stimulated expression of IGFBP1 but not IGFBP3; however, the effect of IFNT did not mimic the abundant increase observed in pregnant ewes. Therefore, IGFBP1 expression in the endometrium is regulated by another factor from the conceptus. IGFBP1 did not affect the proliferation of ovine trophectoderm cells in vitro but did stimulate their migration and mediate their attachment. These studies reveal that IGFBP1 is a common endometrial marker of conceptus elongation in sheep and cattle and most likely regulates conceptus elongation by stimulating migration and attachment of the trophectoderm.

Gastrin-Releasing Peptide (GRP) in the Ovine Uterus: Regulation by Interferon Tau and Progesterone

Abstract Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.

Stanniocalcin 1 Is a Luminal Epithelial Marker for Implantation in Pigs Regulated by Progesterone and Estradiol

Stanniocalcin 1 (STC1) is a glycoprotein that decreases calcium and increases phosphate in cells/tissues. This investigation examined endocrine regulation of STC1 in endometria of pigs during the estrous cycle and pregnancy. STC1 mRNA was present exclusively in luminal epithelium (LE) between d 12 and 15 of the estrous cycle, increased between d 12 and d 20, and was not detectable by d 30 of pregnancy. STC1 protein was also detected in uterine flushings. To determine effects of estrogen and progesterone, pigs were ovariectomized and treated with these hormones alone or together. Progesterone, but not estrogen, induced STC1 in LE. Cotreatment with progesterone and estrogen further stimulated STC1 over progesterone alone. To determine effects of pseudopregnancy, nonpregnant gilts were given daily injections of estradiol benzoate from d 11 to d 14. STC1 was not expressed in LE on d 90 of pseudopregnancy, suggesting that the estradiol given to induce pseudopregnancy and/or long-term exposure to progesterone are required for down-regulation of STC1. To determine effects of long-term progesterone, without effects of estradiol, pigs were ovariectomized on d 12, given daily injections of progesterone through d 39, and hysterectomized on d 40 after estrus. STC1 was expressed in LE of progesterone-treated pigs, suggesting that estrogen is involved in down-regulation of STC1. We conclude that STC1 is induced in LE by progesterone and further stimulated by estrogen, and its down-regulation in LE by d 25 likely requires exposure of the progestinized uterus to estrogen. The temporal and cell type-specific expression of STC1 makes this gene a unique marker for implantation in pigs.

Progesterone and Interferon Tau Regulate Hypoxia-Inducible Factors in the Endometrium of the Ovine Uterus

In ruminants, progesterone (P4) from the ovary and interferon tau (IFNT) from the elongating blastocyst regulate expression of genes in the endometrium that are hypothesized to be important for uterine receptivity and blastocyst development. These studies determined effects of the estrous cycle, pregnancy, P4, and IFNT on hypoxia-inducible factor (HIF) expression in the ovine uterus. HIF1A mRNA, HIF2A mRNA, and HIF2A protein were most abundant in the endometrial luminal and superficial glandular epithelia (LE and sGE, respectively) of the uterus and conceptus trophectoderm. During the estrous cycle, HIF1A and HIF2A mRNA levels were low to undetectable on d 10 in the endometrial LE/sGE, increased between d 10 and 14, and then declined on d 16. Both HIF1A and HIF2A mRNA were more abundant in the endometrial LE/sGE of pregnant ewes. However, HIF3A, HIF1B, HIF2B, and HIF3B mRNA abundance was low in most cell types of the endometria and conceptus. Treatment of ovariectomized ewes with P4 induced HIF1A and HIF2A in the endometrial LE/sGE, and intrauterine infusion of ovine IFNT further increased HIF2A in P4-treated ewes, but not in ewes treated with P4 and the antiprogestin ZK 136,317. HIF3A, HIF1B, HIF2B, and HIF3B mRNA abundance was not regulated by either P4 or IFNT. Two HIF-responsive genes, carboxy-terminal domain 2 and vascular endothelial growth factor A, were detected in both the endometrium and conceptus. These studies identified new P4-induced (HIF1A and HIF2A) and IFNT-stimulated (HIF2A) genes in the uterine LE/sGE, and implicate the HIF pathway in regulation of endometrial epithelial functions and angiogenesis, as well as peri-implantation blastocyst development.

Progesterone and interferon tau regulate leukemia inhibitory factor receptor and IL6ST in the ovine uterus during early pregnancy

The actions of leukemia inhibitory factor (LIF) via LIF receptor (LIFR) and its co-receptor, IL6 signal transducer (IL6ST), are implicated in uterine receptivity to conceptus implantation in a number of species including sheep. The present study determined the effects of the estrous cycle, pregnancy, progesterone (P4), and interferon tau (IFNT) on the expression of LIFR and IL6ST in the ovine uterus. LIFR mRNA and protein were localized to the endometrial luminal (LE) and superficial glandular epithelia (sGE), whereas IL6ST mRNA and protein were localized primarily in the middle to deep GE. Both LIFR and IL6ST mRNAs and protein were more abundant in pregnant than cyclic ewes and increased from days 10 to 20 of pregnancy. Treatment of ovariectomized ewes with P4 and/or infusion of ovine IFNT increased LIFR and IL6ST in endometrial LE/sGE and GE respectively. Co-expression of LIFR and IL6ST as well as phosphorylated STAT3 was observed only in the upper GE of the endometrium as well as in the conceptus trophectoderm on days 18 and 20. In mononuclear trophectoderm and GE cells, LIF elicited an increase in phosphorylated STAT3 and MAPK3/1 MAPK proteins. Collectively, these results suggest that LIFR and IL6ST are both stimulated by IFNT and regulated by P4 in a complex stage- and cell-specific manner, and support the hypothesis that LIF exerts effects on the endometrial GE as well as conceptus trophectoderm during early pregnancy in sheep. Thus, LIF and STAT3 may have biological roles in endometrial function and trophectoderm growth and differentiation.